Elucidating pro-metastatic collagen modifying activities of lysyl hydroxylase 2

阐明赖氨酰羟化酶 2 的促转移胶原蛋白修饰活性

• 批准号: 10208217
• 负责人: Jonathan M Kurie
• 金额: $ 55.42万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10208217
• 关键词: 2-Oxoglutarate 5-Dioxygenase Procollagen-Lysine; Acidic Amino Acids; Active Sites; Address; Affect; Affinity; Aldehydes; Alternative Splicing; Arginine; Basic Amino Acids; Binding; Biological Assay; Cancer Model; Cancer Patient; Cells; Coculture Techniques; Collagen; Collagen Fibril; Collagen Receptors; Crystallization; Electrostatics; Enzymes; Exons; Family member; Fibroblasts; Future; Glucose; Glucosyltransferase; Hydroxylysine; Immune; Immunocompetent; Immunosuppression; KRAS2 gene; Knowledge; Lysine; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Modification; Mus; Myeloid Cells; Natural Killer Cells; Neoplasm Metastasis; Population; Production; Property; Protein Isoforms; RNA Splicing; Receptor Activation; Reporting; Rest; Source; Specific qualifier value; Structure; Structure-Activity Relationship; T-Lymphocyte; Testing; The Cancer Genome Atlas; Tumor Immunity; base; cancer cell; cell behavior; cohort; crosslink; differential expression; efficacy testing; glycosyltransferase; high throughput screening; insight; lung cancer cell; mechanical force; migration; mutant; novel therapeutics; tool; tumor; tumor progression; tumor-immune system interactions

As they progress, malignant tumors accumulate cross-linked collagen fibrils that enhance matrix stiffness, thereby activating collagen receptors that trigger cancer cell invasion and dissemination. We previously reported that a collagen modifying enzyme called lysyl hydroxylase 2 (LH2) is highly expressed in metastatic lung cancer cells and promotes metastasis by increasing the amount of a particularly stable type of collagen cross-link called hydroxylysine aldehyde-derived collagen cross-link (HLCC). Although LH family members (LH1-3) have highly conserved LH and glucosylgalactosyltransferase (GGT) domains, LH2 reportedly lacks GGT activity and is unique among LHs in its ability to hydroxylate lysine (lys) residues on collagen N- and C- termini (“telopeptides”) that are required to generate HLCCs. The structural basis for LH2's unique functional properties remains unclear. The studies proposed herein will address this crucial knowledge gap. On the basis of collagen LH and GGT domain crystal structures that we recently solved, we show that LH2 has telopeptidyl LH (t-LH) activity owing to a unique basic residue cluster that generates electrostatic interactions with acidic residues on collagen. Furthermore, by using a new collagen GGT activity assay we developed that is more sensitive than ones reported previously, we show that LH2 has GGT activity owing to an alternatively spliced exon 13a-encoded loop that resides at the entrance of the GGT active site. We show that LH2 isoforms that lack (LH2a) or contain (LH2b) exon 13a are differentially expressed in the TCGA lung cancer cohort, and that LH2b is the predominant isoform expressed in an orthotopic KMLC model in which LH2 promotes metastasis and causes widespread alterations in intra-tumoral immunity. We developed defined collagen matrices that are deficient or replete in LH2-mediated HLCCs (total or glucosylated) and used these matrices as tools to show that HLCCs influence lung cancer cell behaviors. On the basis of these preliminary results, we postulate that LH2 drives lung cancer metastasis through dual (LH- and GGT-mediated) collagen modifications and will test this hypothesis by completing 3 specific aims. Aim 1: To demonstrate a causal relationship between LH2's electrostatic interactions with collagen, HLCC formation, and lung cancer metastasis. Aim 2: To demonstrate a causal relationship between inclusion of LH2's exon 13a, collagen glucosylation, and metastasis. Aim 3: To demonstrate a causal relationship between LH2's dual (t-LH- and GGT-mediated) collagen modifications and LAIR-1-mediated immunosuppression. In summary, the novelty of our proposal rests in an hypothesis that is based on unique insight into LH2's dual enzymatic activities and the tools we developed to generate that hypothesis (e.g., crystal structures, enzymatic assays, and defined collagen matrices). Findings from these studies will provide a basis for future testing of selective LH2 antagonists that we have already identified from high-throughput screens and have begun to optimize outside the scope of this application.

随着肿瘤的进展，恶性肿瘤会积累交联的胶原纤维，从而增强基质的硬度， 从而激活胶原蛋白受体，引发癌细胞侵袭和扩散。我们之前 据报道，一种称为赖氨酰羟化酶 2 (LH2) 的胶原蛋白修饰酶在转移性肿瘤中高度表达。 通过增加特别稳定类型的胶原蛋白的量来抑制肺癌细胞并促进转移 这种交联称为羟赖氨酸醛衍生胶原交联（HLCC）。虽然LH家族成员 (LH1-3) 具有高度保守的 LH 和葡萄糖基半乳糖基转移酶 (GGT) 结构域，据报道 LH2 缺乏 GGT 活性，并且在 LH 中独一无二，能够羟基化胶原蛋白 N- 和 C- 上的赖氨酸 (lys) 残基 生成 HLCC 所需的末端（“端肽”）。 LH2独特功能的结构基础 属性仍不清楚。本文提出的研究将解决这一关键的知识差距。在此基础上 根据我们最近解决的胶原蛋白 LH 和 GGT 结构域晶体结构，我们发现 LH2 具有端肽基 LH (t-LH) 活性归因于独特的碱性残基簇，可与酸性物质产生静电相互作用 胶原蛋白上有残留。此外，通过使用新的胶原蛋白 GGT 活性测定，我们开发了更多 比之前报道的敏感，我们表明 LH2 由于选择性剪接而具有 GGT 活性 外显子 13a 编码的环位于 GGT 活性位点的入口处。我们证明 LH2 同工型 缺乏 (LH2a) 或包含 (LH2b) 外显子 13a 在 TCGA 肺癌队列中存在差异表达，并且 LH2b 是原位 KMLC 模型中表达的主要亚型，其中 LH2 促进转移 并导致肿瘤内免疫的广泛改变。我们开发了明确的胶原蛋白基质 LH2 介导的 HLCC（全部或糖基化）缺乏或充足，并使用这些矩阵作为工具来显示 HLCC 影响肺癌细胞的行为。根据这些初步结果，我们假设 LH2 通过双重（LH 和 GGT 介导）胶原蛋白修饰驱动肺癌转移，并将测试 通过完成 3 个具体目标来实现这一假设。目标 1：证明 LH2 之间的因果关系 与胶原蛋白的静电相互作用、HLCC 形成和肺癌转移。目标 2：展示 LH2 外显子 13a 的包含、胶原糖基化和转移之间的因果关系。目标 3： 证明 LH2 的双重（t-LH 和 GGT 介导的）胶原蛋白修饰之间的因果关系 LAIR-1 介导的免疫抑制。总之，我们提案的新颖性在于一个假设： 基于对 LH2 双酶活性的独特见解以及我们开发的工具来生成该活性 假设（例如晶体结构、酶测定和确定的胶原蛋白基质）。从这些结果中得出的结论 研究将为未来测试我们已经鉴定出的选择性 LH2 拮抗剂提供基础 高通量筛选，并已开​​始在此应用范围之外进行优化。